• Tuberculosis and Respiratory Diseases
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• v.41 no.4
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• pp.319-327
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• 1994

Background: The pathogenetic mechanism of adult respiratory distress syndrome(ARDS) is not clearly defined yet, but it is well known that increased pulmonary capillary permeabilty is characteristic feature of ARDS. The increased alveolar-capillary permeability is usually preceded by damage of pulmonary artery endothelial cells. The released enzymes and oxygen free radicals from the activated neutrophils seem to play a predominant role in endothelial cell cytotoxicity. The activated neutrophils, however, probably are not the sole contributing factor in this type of damage because many cases of ARDS have been reported in severe neutropenia. Bacterial endotoxin perse and/or oxygen free radicals released from endothelial cells are suggested to be possible factors that contribute to the development of ARDS. The purpose of this study is to investigate the direct cytotoxicity of endotoxin and the role of oxygen free radicals released from the endothelial cells in endotoxin-induced endothelial cell cytotoxicity. Methods: First, to investigate whether endotoxin is cytotoxic to HUVE by itself, various doses of endotoxin were added to culture medium and cytotoxicity was measured. Second, to evaluate the possible role of oxygen free radical in endotoxin-induced HUVE cytotoxicity, various antioxidants were added on the endotoxin-induced HUVE cytotoxicity and cytotoxicity was measured. Third, to verify the release of oxygen free radicals from HUVE, the concentrations of hydrogen peroxide in the endotoxin-treated culture supernatant were measured. Finally, to observe the cytotoxic effect of hydrogen peroxide, HUVE cytotoxicity in the presence of various doses of hydrogen peroxide was measured. The fourth generations of subcultured HUVE from primary culture were used. The cell cytotoxicity was quantified by the chromium-51 release assay. Results: 1) Endotoxin alone showed HUVE cytotoxicity in a dose-dependent fashion. 2) Endotoxin-induced HUVE cytotoxicity was significantly attenuated by the pretreatment of catalase and DMTU. 3) Hydrogen peroxide was released from HUVE after endotoxin treatment in a dose-dependent fashion. 4) Exogenous hydrogen peroxide also showed HUVE cytotoxicity in a dose-dependent fashion. Conclusion: These results suggest that endotoxin alone can directly injure HUVE, and, oxygen-free radicals released from HUVE in response to endotoxin may also participate in the endotoxin-induced HUVE cytotoxicity.

• Tuberculosis and Respiratory Diseases
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• v.38 no.4
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• pp.357-371
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• 1991

To identify the pathogenetic role of reactive oxygen free radical-induced oxidation reaction in endotoxin-induced acute lung injury, we infused endotoxin into 8 domestic pigs; endotoxin only (n=3), pretreatment with dimethylthiourea (DMTU) (n=5). We observed the sequential changes in hemodynamic parameters, the concentration of plasma oxidized glutathione (GSSG) in pulmonary arterial and venous blood, and albumin content in bronchoalveolar lavage fluid (BALF). The results were as follows. 1) While cardiac output decreased, mean pulmonary arterial pressure, pulmonary vascular resistance, and alveolar-arterial oxygen difference increased over phase 1 (0-2 hr) and phase 2 (2-4.5 hr) by endotoxin, DMTU attenuated the above changes only during phase 2. 2) While the concentration of plasma GSSG increased significantly by endotoxin during phase 2, there were no significant differences between pulmonary arterial and venous GSSG contents during both phases. The increase in plasma GSSG content was attenuated by DMTU. 3) The content of BALF albumin was significantly lower in DMTU group than that of endotoxin group. These results suggest that reactive oxygen free radical-induced oxidation reaction may have an important pathogenetic role in endotoxin-induced acute lung injury in pigs, which seems to be greater during phase 2 rather than phase 1.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.554-558
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• 2015

Drug delivery systems (DDSs) incorporating bacterial minicells have been evaluated as a very powerful tool in view of biocompatibility. However, limited studies have been carried out on these systems, mainly using minicells from Salmonella sp. and Escherichia coli. Thus, we generated a new minicell-producing strain from an endotoxin-free Corynebacterium glutamicum by the inactivation of genes related to cell division. The two knockout strains, ${\Delta}parA$ and ${\Delta}ncgl1366$, showed distinct abilities to produce minicells. The resulting minicells were purified via sequential antibiotic treatments and centrifugations, which resulted in reproducible yields.

• Biomolecules & Therapeutics
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• v.8 no.3
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• pp.223-227
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• 2000

Propolis, a natural resinous compound collected from honey bees, contains many biochemical constituents and has been used for traditional medicines as early as 300 B .C. Recently, it has been reported to possess many biological activities such as antibacterial, antiviral, fungicidal, local anaesthetic, immunostimulating, antiinflammatory and free radical scavenging properties. To investigate activities of chrysin, one of propolis effective compounds for blood coagulation system was injected endotoxin (4000 EU/kg, i.v.) in rats at 1 hr after administered chrysin (20 mg/kg, p.o.). This study was resulted that chrysin has antiplatelet aggregation activity in vitro, delay of blood clotting time and prothrombin time, and reduction of fibrinogen and FDP in vivo. Chrysin has increased SOD activity, GSH content and GST activity, and decreased MDA content in liver. The result suggests that the antithrombosis effect of chrysin is suppressive activity for a blood coagulation system and antioxidative activity.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Tuberculosis and Respiratory Diseases
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• v.40 no.3
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• pp.223-235
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• 1993

Background: Superoxide anion which was produced by macrophage and neutrophil has a defensive role to kill invasive microorganisms and also an injurious role to produce self lung damage. Production of oxygen free radicals including superoxide is a main mechanism of acute lung injury caused by bacterial endotoxin. Endotoxin is known to activate alveolar macrophage to produce increased oxygen free radicals after the stimulation with various biological materials (priming effect). Calcium is a very important intracellular messenger in that cellular process of superoxide production. Method: This experiment was performed to elucidate the effects of endotoxin and calcium on superoxide production by phorbol myristate acetate-stimulated alveolar macrophage and the effect of verapamil on priming effect of endotoxin. Results: 1) Preincubation of macrophages with endotoxin (E. coli 055-B5) primed the cells to respond with increased superoxide production after the stimulation with PMA. Priming with endotoxin ($10^{-1}$ug/ml) produced a maximal enhancement of superoxide production (43%). 2) Verapamil could inhibit the superoxide production by PMA stimulated macrophage regardless of the presence of extracellular calcium. This means that the inhibitory effect of verapamil is caused by a mechanism independent of blocking calcium influx. 3) Verapamil could inhibit the priming effect of endotoxin on alveolar macrophage (from 30% increment to 13% increment) and could inhibit the superoxide production by PMA-stimulated macrophage preincubated with endotoxin. Conclusion: We concluded that verapamil could inhibit the superoxide production by PMA-stimulated rat alveolar macrophage and also inhibit the priming effect of endotoxin on alveolar macrophage. These inhibitory effects of verapamil could be one of the mechanisms of verapamil effects on endotoxin induced lung injury.

• Tuberculosis and Respiratory Diseases
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• v.39 no.2
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• pp.120-130
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• 1992

Background: Although there have been many studies on the pathogenetic mechanism of acute lung injury, it is still elusive. Recently interests have been focused on the role of oxygen free radicals. But the effect of hydroxyl radical on the neutrophil mobilization and the alveolar-capillary permeability is not clear especially in the endotoxin-induced acute lung injury model of rats. This investigation was performed to evaluate the pathogenetic role of hydroxyl radical on the neutrophil accumulation into the lung and the increased alveolar-capillary permeability in the endotoxin-induced acute lung injury in rats. Method: Fifty rats were divided into four groups: vehicle control group (n=5, 6hrs; n=5, 24hrs), endotoxin-treated group (n=10, 6hrs; n=10, 24hrs), Dimethylthiourea (DMTU)-pretreated group (n=10, 6hrs), and deferoxamine (DFX)-pretreated group (n=10, 6hrs). Thirty minutes before sacrifice, $^{125}I$-tagged bovine serum albumin was injected. Six and twenty four hours after endotoxin injection, the rats were sacrificed, and the radioactivity of lung tissue and peripheral blood was counted. Permeability index was defined as the ratio of radioactivity between lung tissue and peripheral blood. Another set of rats (n=52) were divided into the same four groups as before [vehicle control group (n=5, 6hrs; n=5, 24hrs), endotoxin·treated group (n=7, 6hrs; n=8, 24hrs), DMTU-pretreated group (n=6, 6hrs; n=9, 24hrs), and DFX-pretreated group (n=5, 6hrs; n=7, 24hrs)], and were sacrificed 6 and 24 hours after endotoxin injection. In these rats, cell profile of peripheral blood and bronchoalveolar lavage fluid was evaluated, and the pathologic examination of lung tissue was performed. Results: 1) Increased alveolar-capillary permeability was observed 6 hours after endotoxin injection, which was normalized after 24 hours, and this increase was attenuated by pretreatment with DMTU and DFX. 2) Neutrophil sequestration into the lung was observed 24 hours after endotoxin administration, but this was not influenced by DMTU and DFX pretreatment. Conclusion: These results suggest that hydroxyl radical would not be involved in the sequestration of neutrophils into the lung, but plays an important role in the increase of alveolar-capillary permeability in the endotoxin-induced acute lung injury in rats.

• Tuberculosis and Respiratory Diseases
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• v.39 no.1
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• pp.42-54
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• 1992

Background:It has been suggested that the cyclooxygenase metabolites play an important role in changes of early hemodynamic parameters in the endotoxin-induced acute lung injury. But there have been many debates about their role in the late increase of alveolar-capillary permeability, and it is not known whether they act directly or indirectly through oxygen free radicals which have been known to be produced during the metabolic process of cyclooxygenase pathway. So we performed this study to identify the pathogenetic role of cyclooxygenase metabolites in the endotoxin-induced acute lung injury in domestic pigs. Method: We infused endotoxin into 8 domestic pigs; endotoxin only (n=3), and pretreatment with indomethacin (n=5). We observed the sequential changes in hemodynamic parameters, the concentration of plasma oxidized glutathione (GSSG) in pulmonary arterial and venous blood, and albumin content in bronchoalveolar lavage fluid (BALF). Results: 1) While cardiac output decreased, mean pulmonary arterial pressure, pulmonary vascular resistance, and alveolar-arterial oxygen difference increased over phase 1 (0-2hr) and phase 2 (2-4.5hr) by endotoxin, indomethacin attenuated the decrease in cardiac output during phase 1 and increase in mean pulmonary arterial pressure, pulmonary vascular resistance, and alveolar-arterial oxygen difference during both phases. 2) The increase in plasma GSSG content during phase 2 was not attenuated by indomethacin. 3) The content of BALF albumin was significantly lower in indomethacin groups than that of endotoxin group. Conclusion: These results suggest that it is likely that cyclooxygenase metabolites have an effect on endotoxin-induced acute lung injury during both phases probably through direct action.

• Journal of Periodontal and Implant Science
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• v.34 no.1
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• pp.71-81
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• 2004

Peri-implantitis could be the result of biomechanical and occlusal overload as well as microbiologic invasion. The dental implant may be more susceptible to dental plaque than the natural tooth, as the predictability of a stable soft tissue attachment complex has not yet been confirmed. With the development of peri-implantitis, the implant surface would be exposed to the oral environment and becomes coated with bacteria. The objective of therapy for this condition is to regain integration of the implant with bone. Since fibroblast adherence to surfaces is impeded by endotoxin, it would seem that decontamination would be desirable to obtain maximum osseointegration. The purpose of this study was to determine whether various chemotherapeutic and mechanical treatments(distilled water, air-powder abrasive, hypersaturated citric acid, tetracycline) can detoxify contaminated titanium implant surface by means of kinetic LAL test. Experimental rough surface titanium disks were fabricated. All of them were divided into two groups(A.a group and P.g group) and each contaminated by A. actinomycetemcomitans and P. gingivalis suspension. Contaminated disks were treated with distilled water, air-powder abrasive, citric acid and tetracycline, and then all disks were placed into LPS-free water for elution. The results were as follows : 1. In A.a group, LPS elute level of all test groups were significantly lower than control group(p<0.05). 2. In A.a group, LPS elute level of test 2, test 3 and test4 groups were significantly lower than that of control group(p<0.05). But, among the test 2, test 3, test4 groups, the significant differences were not detected. 3. In P.g group, LPS elute level of test 2, test 3 and test 4 groups were lower than that of control group(p<0.05). But, among the test groups, the significant differences were not detected. From the result of this study, it would be concluded that air-powder abrasive, hypersaturated citric acid and tetracycline treatments may be effective at reducing endotoxin level on rough titanium implant surfaces, and can be clinically used. But the treatments in peri-implantitis differentially impact osseointegration making one method clinically superior. To gain this knowledges, further molecular biologic and histopathologic studies should be developed.

• Tuberculosis and Respiratory Diseases
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• v.60 no.4
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• pp.437-450
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• 2006

Background : Acute respiratory distress syndrome (ARDS) is characterized by severe inflammatory pulmonary edema of unknown pathogenesis. To investigate the pathogenesis of ARDS associated with neutrophilic oxidative stress, the role of phospholipase $A_2$ ($PLA_2$) was evaluated by the inhibition of calcium channel. Methods : In Sprague-Dawley rats, acute lung injury (ALI) was induced by the instillation of E.coli endotoxin (ETX) into the trachea. At the same time, diltiazem was given 60 min prior to tracheal instillation of ETX. Parameters of ALI such as lung and neutrophil $PLA_2$, lung myeloperoxidase (MPO), BAL neutrophils, protein, surfactant were measured. Production of free radicals from neutrophils was measured also. Morphological studies with light microscope and electron microscope were carried out and electron microscopic cytochemistry for detection of free radicals was performed also. Results : Diltiazem had decreased the ALI parameters effectively in ETX given rats and decreased the production of free radicals from neutrophils and lung tissues. Morphological studies denoted the protective effects of diltiazem. Conclusion : Diltiazem, a calcium channel blocker, was effective in amelioration of ALI by the suppression of neutrophilic oxidative stress mediated by $PLA_2$ activation.